Succinic acid production in a eukaryotic cell

ABSTRACT

The present invention relates to a recombinant eukaryotic cell selected from a yeast of a filamentous fungus comprising a nucleotide sequence encoding a NAD(H)-dependent fumarate reductase that catalyses the conversion of fumaric acid to succinic acid. The invention further relates to a process for the production of succinic acid wherein the eukaryotic cell according to the present invention is used.

The present invention relates to a recombinant eukaryotic cellcomprising a nucleotide sequence encoding a fumarate reductase and aprocess for the production of succinic acid wherein the recombinanteukaryotic cell is used.

Succinic acid is a potential precursor for numerous chemicals. Forexample, succinic acid can be converted into 1,4-butanediol (BDO),tetrahydrofuran, and gamma-butyrolactone. Another product derived fromsuccinic acid is a polyester polymer which is made by linking succinicacid and BDO.

Succinic acid is predominantly produced through petrochemical processesby hydrogenation of butane. These processes are considered harmful forthe environment and costly. The fermentative production of succinic acidmay be an attractive alternative process for the production of succinicacid, wherein renewable feedstock as a carbon source may be used.

A number of different bacteria such as Escherichia coli, and the rumenbacteria Actinobacillus, Anaerobiospirillum, Bacteroides, Mannheimia, orSuccinimonas, sp. are known to produce succinic acid. Metabolicengineering of these bacterial strains have improved the succinic acidyield and/or productivity, or reduced the by-product formation.

WO2007/061590 discloses a pyruvate decarboxylase negative yeast for theproduction of malic acid and/or succinic acid which is transformed witha pyruvate carboxylase enzyme or a phosphoenolpyruvate carboxylase, amalate dehydrogenase enzyme, and a malic acid transporter protein (MAE).

Despite the improvements that have been made in the fermentativeproduction of succinic acid, there remains a need for improvedmicroorganisms for the fermentative production of succinic acid.

The aim of the present invention is an alternative microorganism for theproduction of succinic acid.

The aim is achieved according to the invention with a recombinanteukaryotic cell selected from the group consisting of a yeast and afilamentous fungus comprising a nucleotide sequence encodingNAD(H)-dependent fumarate reductase that catalyses the conversion offumaric acid to succinic acid.

Surprisingly it was found that the recombinant eukaryotic cell accordingto the present invention produces an increased amount of succinic acidcompared to the amount of succinic acid produced by a wild-typeeukaryotic cell. Preferably, a eukaryotic cell according to the presentinvention produces at least 1.2, preferably at least 1.5, preferably atleast 2 times more succinic acid than a wild-type eukaryotic cell whichdoes not comprise the nucleotide sequence encoding NAD(H)-dependentfumarate reductase.

As used herein, a recombinant eukaryotic cell according to the presentinvention is defined as a cell which contains, or is transformed orgenetically modified with a nucleotide sequence or polypeptide that doesnot naturally occur in the eukaryotic cell, or it contains additionalcopy or copies of an endogenous nucleic acid sequence. A wild-typeeukaryotic cell is herein defined as the parental cell of therecombinant cell.

The nucleotide sequence encoding a NAD(H)-dependent fumarate reductasethat catalyses the conversion of fumaric acid to succinic acid may be aheterologous or homologous nucleotide sequence, or encodes aheterologous or homologous NAD(H)-dependent fumarate reductase, whichmay have been further genetically modified by mutation, disruption ordeletion. Recombinant DNA techniques are well known in the art such asin Sambrook and Russel (2001) “Molecular Cloning: A Laboratory Manual(3^(rd) edition), Cold Spring Harbor Laboratory Press.

The term “homologous” when used to indicate the relation between a given(recombinant) nucleic acid or polypeptide molecule and a given hostorganism or host cell, is understood to mean that in nature the nucleicacid or polypeptide molecule is produced by a host cell or organisms ofthe same species, preferably of the same variety or strain.

The term “heterologous” when used with respect to a nucleic acid (DNA orRNA) or protein refers to a nucleic acid or protein that does not occurnaturally as part of the organism, cell, genome or DNA or RNA sequencein which it is present, or that is found in a cell or location orlocations in the genome or DNA or RNA sequence that differ from that inwhich it is found in nature. Heterologous nucleic acids or proteins arenot endogenous to the cell into which it is introduced, but have beenobtained from another cell or synthetically or recombinantly produced.

A NAD(H)-dependent fumarate reductase according to the present inventionuses NAD(H) as a cofactor, whereas most eukaryotic cells comprise aFADH₂-dependent fumarate reductase, wherein FADH₂ is the cofactor. Itwas found advantageous that the eukaryotic cell comprises a nucleotidesequence encoding a NAD(H)-dependent fumarate reductase, since theNAD(H)-dependent fumarate reductase provides the cell with furtheroptions to oxidise NAD(H) to NAD⁺ and influence the redox balance in thecell.

Preferably, the cell expresses a nucleotide sequence encoding an enzymethat catalyses the formation of succinic acid, wherein the nucleotidesequence preferably encodes a NAD(H)-dependent fumarate reductase,comprising an amino acid sequence that has at least 40%, preferably atleast 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, 99% sequenceidentity with the amino acid sequence of SEQ ID NO: 1, and/or SEQ ID NO:3, and/or SEQ ID NO: 4, and/or SEQ ID NO: 6. Preferably, the nucleotidesequence encodes a NAD(H)-dependent fumarate reductase comprising theamino acid sequence of SEQ ID NO: 1, and/or SEQ ID NO: 3, and/or SEQ IDNO: 4, and/or SEQ ID NO: 6.

Sequence identity is herein defined as a relationship between two ormore amino acid (polypeptide or protein) sequences or two or morenucleic acid (polynucleotide) sequences, as determined by comparing thesequences. Usually, sequence identities or similarities are comparedover the whole length of the sequences compared. In the art, “identity”also means the degree of sequence relatedness between amino acid ornucleic acid sequences, as the case may be, as determined by the matchbetween strings of such sequences.

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include BLASTP and BLASTN, publicly available fromNCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIHBethesda, Md. 20894). Preferred parameters for amino acid sequencescomparison using BLASTP are gap open 11.0, gap extend 1, Blosum 62matrix.

Nucleotide sequences encoding the enzymes expressed in the cell of theinvention may also be defined by their capability to hybridise with thenucleotide sequences encoding a NAD(H) dependent fumarate reductase ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, and/or SEQ ID NO: 6, undermoderate, or preferably under stringent hybridisation conditions.Stringent hybridisation conditions are herein defined as conditions thatallow a nucleic acid sequence of at least about 25, preferably about 50nucleotides, 75 or 100 and most preferably of about 200 or morenucleotides, to hybridise at a temperature of about 65° C. in a solutioncomprising about 1 M salt, preferably 6×SSC (sodium chloride, sodiumcitrate) or any other solution having a comparable ionic strength, andwashing at 65° C. in a solution comprising about 0.1 M salt, or less,preferably 0.2×SSC or any other solution having a comparable ionicstrength. Preferably, the hybridisation is performed overnight, i.e. atleast for 10 hours and preferably washing is performed for at least onehour with at least two changes of the washing solution. These conditionswill usually allow the specific hybridisation of sequences having about90% or more sequence identity.

Moderate conditions are herein defined as conditions that allow anucleic acid sequences of at least 50 nucleotides, preferably of about200 or more nucleotides, to hybridise at a temperature of about 45° C.in a solution comprising about 1 M salt, preferably 6×SSC or any othersolution having a comparable ionic strength, and washing at roomtemperature in a solution comprising about 1 M salt, preferably 6×SSC orany other solution having a comparable ionic strength. Preferably, thehybridisation is performed overnight, i.e. at least for 10 hours, andpreferably washing is performed for at least one hour with at least twochanges of the washing solution. These conditions will usually allow thespecific hybridisation of sequences having up to 50% sequence identity.The person skilled in the art will be able to modify these hybridisationconditions in order to specifically identify sequences varying inidentity between 50% and 90%.

To increase the likelihood that an introduced enzyme(s) is/are expressedin active form in a eukaryotic cell of the invention, the correspondingencoding nucleotide sequence may be adapted to optimise its codon usageto that of the chosen eukaryote host cell. Several methods for codonoptimisation are known in the art. A preferred method to optimise codonusage of the nucleotide sequences to that of the eukaryotic cell is acodon pair optimization technology as disclosed in WO2008/000632.Codon-pair optimization is a method for producing a polypeptide in ahost cell, wherein the nucleotide sequences encoding the polypeptidehave been modified with respect to their codon-usage, in particular thecodon-pairs that are used, to obtain improved expression of thenucleotide sequence encoding the polypeptide and/or improved productionof the polypeptide. Codon pairs are defined as a set of two subsequenttriplets (codons) in a coding sequence.

The term “gene”, as used herein, refers to a nucleic acid sequencecontaining a template for a nucleic acid polymerase, in eukaryotes, RNApolymerase II. Genes are transcribed into mRNAs that are then translatedinto protein.

The term “nucleic acid” as used herein, includes reference to adeoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, ineither single- or double-stranded form, and unless otherwise limited,encompasses known analogues having the essential nature of naturalnucleotides in that they hybridize to single-stranded nucleic acids in amanner similar to naturally occurring nucleotides (e.g., peptide nucleicacids). A polynucleotide can be full-length or a subsequence of a nativeor heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. The terms“polypeptide”, “peptide” and “protein” are also inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation.

The term “enzyme” as used herein is defined as a protein which catalysesa (bio)chemical reaction in a cell.

Usually, the nucleotide sequence encoding an enzyme is operably linkedto a promoter that causes sufficient expression of the correspondingnucleotide sequence in the eukaryotic cell according to the presentinvention to confer to the cell the ability to produce succinic acid.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements (or coding sequences or nucleic acid sequence)in a functional relationship. A nucleic acid sequence is “operablylinked” when it is placed into a functional relationship with anothernucleic acid sequence. For instance, a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thecoding sequence.

As used herein, the term “promoter” refers to a nucleic acid fragmentthat functions to control the transcription of one or more genes,located upstream with respect to the direction of transcription of thetranscription initiation site of the gene, and is structurallyidentified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites and any other DNA sequencesknown to one of skilled in the art. A “constitutive” promoter is apromoter that is active under most environmental and developmentalconditions. An “inducible” promoter is a promoter that is active underenvironmental or developmental regulation.

A promoter that could be used to achieve the expression of a nucleotidesequence coding for an enzyme such as NAD(H)-dependent fumaratereductase or any other enzyme introduced in the eukaryotic cell of theinvention, may be not native to a nucleotide sequence coding for theenzyme to be expressed, i.e. a promoter that is heterologous to thenucleotide sequence (coding sequence) to which it is operably linked.Preferably, the promoter is homologous, i.e. endogenous to the hostcell.

Suitable promoters in this context include both constitutive andinducible natural promoters as well as engineered promoters, which arewell known to the person skilled in the art. Suitable promoters ineukaryotic host cells may be GAL7, GAL10, or GAL 1, CYC1, HIS3, ADH1,PGL, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, and AOX1. Othersuitable promoters include PDC, GPD1, PGK1, TEF1, and TDH.

Usually a nucleotide sequence encoding an enzyme comprises a terminator.Any terminator, which is functional in the eukaryotic cell, may be usedin the present invention. Preferred terminators are obtained fromnatural genes of the host cell. Suitable terminator sequences are wellknown in the art. Preferably, such terminators are combined withmutations that prevent nonsense mediated mRNA decay in the host cell ofthe invention (see for example: Shirley et al., 2002, Genetics161:1465-1482).

In a preferred embodiment, a nucleotide sequence encoding aNAD(H)-dependent fumarate reductase may be overexpressed to achieve asufficient production of succinic acid by the cell.

There are various means available in the art for overexpression ofnucleotide sequences encoding enzymes in a eukaryotic cell of theinvention. In particular, a nucleotide sequence encoding an enzyme maybe overexpressed by increasing the copy number of the gene coding forthe enzyme in the cell, e.g. by integrating additional copies of thegene in the cell's genome, by expressing the gene from a centromericvector, from an episomal multicopy expression vector or by introducingan (episomal) expression vector that comprises multiple copies of thegene. Preferably, overexpression of the enzyme according to theinvention is achieved with a (strong) constitutive promoter.

The invention also relates to a nucleotide construct comprising one ormore nucleotide sequence(s) selected from the group consisting of SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

The nucleic acid construct may be a plasmid, for instance a low copyplasmid or a high copy plasmid. The eukaryotic cell according to thepresent invention may comprise a single, but preferably comprisesmultiple copies of the nucleotide sequence encoding a NAD(H) dependentfumarate reductase, for instance by multiple copies of a nucleotideconstruct.

The nucleic acid construct may be maintained episomally and thuscomprise a sequence for autonomous replication, such as an autosomalreplication sequence. If the eukaryotic cell is of fungal origin, asuitable episomal nucleic acid construct may e.g. be based on the yeast2μ or pKD1 plasmids (Gleer et al., 1991, Biotechnology 9: 968-975), orthe AMA plasmids (Fierro et al., 1995, Curr Genet. 29:482-489).Alternatively, each nucleic acid construct may be integrated in one ormore copies into the genome of the eukaryotic cell. Integration into thecell's genome may occur at random by non-homologous recombination butpreferably, the nucleic acid construct may be integrated into the cell'sgenome by homologous recombination as is well known in the art.

The nucleotide sequence encoding a NAD(H)-dependent fumarate reductase,may be a heterologous or a homologous nucleotide sequence. Preferably,the NADH-dependent fumarate reductase is a heterologous enzyme, whichmay be derived from any suitable origin, for instance bacteria, fungi,protozoa or plants. Preferably, the cell according to the inventioncomprises hetereologous a NAD(H)-dependent fumarate reductase,preferably derived from a Trypanosoma sp, for instance a Trypanosomabrucei.

In a preferred embodiment the nucleotide sequence encoding aNAD(H)-dependent fumarate reductase is expressed in the cytosol.Surprisingly, cytosolic activity of the enzyme resulted in an increasedproductivity of succinic acid by the eukaryotic cell.

In the event that the nucleotide sequence encoding a NAD(H)-dependentfumarate reductase comprises a peroxisomal or mitochondrial targetingsignal, it may be essential to modify or delete a number of amino acids(and corresponding nucleotide sequences in the encoding nucleotidesequence) in order to prevent peroxisomal or mitochondrial targeting ofthe enzyme. The presence of a peroxisomal targeting signal may forinstance be determined by the method disclosed by Schluter et al,Nucleic acid Research 2007, 35, D815-D822.

Preferably, the NAD(H)-dependent fumarate reductase lacks a peroxisomalor mitochondrial targeting signal for cytosolic activity of the enzymeupon expression of the encoding nucleotide sequence.

Preferably, the cell expresses a nucleotide sequence encoding an enzymethat catalyses the formation of succinic acid, wherein the nucleotidesequence preferably encodes a NAD(H)-dependent fumarate reductase,preferably a fumarate reductase comprising an amino acid sequence thathas at least 40%, preferably at least 45, 50, 55, 60, 65 70, 75, 80, 85,90, 95, 97, 98, 99% sequence identity with the amino acid sequence ofSEQ ID NO: 3, and/or SEQ ID NO: 6. Preferably the nucleotide sequenceencodes a NAD(H)-dependent fumarate reductase comprising the amino acidsequence of SEQ ID NO: 3, and/or SEQ ID NO: 6.

The eukaryotic cell selected from the group consisting of a yeast and afilamentous fungus, preferably belongs to one of the generaSaccharomyces, Aspergillus, Penicillium, Pichia, Kluyveromyces,Yarrowia, Candida, Hansenula, Humicola, Rhizopus, Torulaspora,Trichosporon, Brettanomyces, Zygosaccharomyces, Pachysolen orYamadazyma. More preferably, the eukaryotic cell is a Saccharomycescervisiae, Saccharomyces uvarum, Saccharomyces bayanus, Aspergillusniger, Penicillium chrysogenum, Pichia stipidis, Kluyveromycesmarxianus, K. lactis, K. thermotolerans, Yarrowia lipolytica, Candidasonorensis, C. glabrata, Hansenula polymorpha, Torulaspora delbrueckii,Brettanomyces bruxellensis, Rhizopus orizae or Zygosaccharomycesbailiff.

In addition to a nucleotide sequence encoding a NAD(H)-dependentfumarate reductase that catalyses the conversion of fumaric acid tosuccinic acid, recombinant eukaryotic cell according to the presentinvention may comprise further genetic modifications, for instancemutations, deletions or disruptions, in homologous nucleotide sequencesand/or transformation with nucleotide sequences that encode homologousor heterologous enzymes that catalyse a reaction in the cell resultingin an increased flux towards succinic acid. It may for example befavourable to introduce, genetically modify and/or overexpressheterologous and/or homologous nucleotide sequences encoding i) anenzyme that catalyses the conversion of phosphoenolpyruvate or pyruvateto oxaloacetate; ii) a malate dehydrogenase which catalyses theconversion from OAA to malic acid; or iii) a fumarase, which catalysesthe conversion of malic acid to fumaric acid.

A eukaryotic cell may be transformed or genetically modified with anysuitable nucleotide sequence catalyzing the reaction from a C3 to C4carbon molecule, such as phosphoenolpyruvate (PEP, C3) to oxaloacetate(OAA, C4) and pyruvate (C3) to OAA or malic acid (C3). Suitable enzymesare PEP carboxykinase (EC 4.1.1.49, EC 4.1.1.38) and PEP carboxylase (EC4.1.1.31) which catalyse the conversion of PEP to OAA; pyruvatecarboxylase (EC 6.4.1.1.), that catalyses the reaction from pyruvate toOAA; or malic enzyme (EC 1.1.1.38), that catalyses the reaction frompyruvate to malic acid.

Preferably a eukaryotic cell according to the present inventionoverexpresses a nucleotide sequence encoding a pyruvate carboxylase(PYC), preferably a pyruvate carboxylase that is active in the cytosolupon expression of the nucleotide sequence encoding a PYC, for instancea PYC comprising an amino acid sequence according to SEQ ID NO: 41.Preferably, an endogenous or homologous pyruvate carboxylase isoverexpressed. Surprisingly, it was found that overexpressing anendogenous pyruvate carboxylase resulted in increased succinic acidproduction levels by the eukaryotic cell according to the presentinvention.

In another preferred embodiment, a eukaryotic cell according to thepresent invention further comprises a nucleotide sequence encoding aheterologous PEP carboxykinase (EC 4.1.1.49) catalysing the reactionfrom phosphoenolpyruvate to oxaloacetate. Surprisingly it was found thata eukaryotic cell according to the present invention which furthercomprises a heterologous PEP carboxykinase produced an increased amountof succinic acid as compared to a eukaryotic cell that does not comprisethe heterologous PEP carboxykinase. Preferably, a PEP carboxykinase thatis derived from bacteria, more preferably the enzyme having PEPcarboxykinase activity is derived from Escherichia coli, Mannheimia sp.,Actinobacillus sp., or Anaerobiospirillum sp., more preferablyMannheimia succiniciproducens, Actinobacillus succinogenes, orAnaerobiospirillum succiniciproducens. Preferably, the PEP carboxykinaseis active in the cytosol upon expression of the nucleotide sequenceencoding PEP carboxykinase since it was found that this resulted in anincrease succinic acid production. In one embodiment the PEPcarboxykinase of Actinobacillus succinogenes (PCKa) has been modified toreplace EGY at position 120-122 with a DAF amino acid sequence.Preferably, a eukaryotic cell according to the present inventioncomprises a PEP carboxykinase which has at least 80, 85, 90, 95 or 99%sequence identity with SEQ ID NO: 14 or SEQ ID NO: 17, preferably a PEPcarboxykinase comprising SEQ ID NO: 14 or SEQ ID NO: 17. Surprisingly itwas found that the concomitant (over)expression of a PYC and a PEPcarboxykinase as described herein resulted in at least 1.5 increase insuccinic acid production.

In another preferred embodiment a cell according to the presentinvention further comprises a nucleotide sequence encoding a malatedehydrogenase (MDH) which is active in the cytosol upon expression ofthe nucleotide sequence. A cytosolic MDH may be any suitable homologousor heterologous malate dehydrogenase. The MDH may be a S. cerevisiaeMDH3 or S. cerevisiae MDH1. Preferably, the MDH lacks a peroxisomal ormitochondrial targeting signal in order to localize the enzyme in thecytosol. Alternatively, the MDH is S. cerevisiae MDH2 which has beenmodified such that it is not inactivated in the presence of glucose andis active in the cytosol. It is known that the transcription of MDH2 isrepressed and Mdh2p is degraded upon addition of glucose toglucose-starved cells. Mdh2p deleted for the first 12 amino-terminalamino acids is less-susceptible for glucose-induced degradation (Minardand McAlister-Henn, J. Biol Chem. 1992 Aug. 25; 267(24):17458-64).Preferably, a eukaryotic cell according to the present inventioncomprises a nucleotide sequence encoding a malate dehydrogenase that hasat least 70%, preferably at least 75, 80, 85, 90, 92, 94, 95, 96, 97,98, 99% sequence identity with the amino acid sequence of SEQ ID NO: 19or SEQ ID NO: 21. Preferaby the malate dehydrogenase comprises SEQ IDNO: 19 or SEQ ID NO: 21. Preferably, the activity of malatedehydrogenase is increased by overexpressing the encoding nucleotidesequence by known methods in the art.

Preferably, a eukaryotic cell according to the present invention furthercomprises a nucleotide sequence encoding an enzyme that catalyses theconversion of malic acid to fumaric acid, which may be a heterologous orhomologous enzyme, for instance a fumarase (FUM). A nucleotide sequenceencoding an heterologous enzyme that catalyses the conversion of malicacid to fumaric acid, may be derived from any suitable origin,preferably from microbial origin, preferably from a yeast, for instanceSaccharomyces cerevisiae or a filamentous fungus, for instance Rhizopusoryzae. Preferably, a eukaryotic cell according to the present inventioncomprises a nucleotide sequence encoding a fumarase that has at least70%, preferably at least 75, 80, 85, 90, 92, 94, 95, 96, 97, 98, or 99%sequence identity with the amino acid sequence of SEQ ID NO: 23.Preferably, the fumarase comprises SEQ ID NO: 23. Preferably the enzymehaving fumarase activity is active in the cytosol upon expression of thenucleotide sequence encoding the enzyme having fumarase activity.Surprisingly, it was found that a eukaryotic cell further comprising anenzyme having fumarase activity as described herein produced anincreased amount of succinic acid.

In another embodiment, a eukaryotic cell according to the presentinvention comprises a nucleotide sequence encoding a dicarboxylic acidtransporter protein, preferably a malic acid transporter protein (MAE).A dicarboxylic acid transporter protein may be a homologous orheterologous protein. Preferably the dicarboxylic acid transporterprotein is a heterologous protein. A dicarboxylic acid transporterprotein may be derived from any suitable organism, preferably fromSchizosaccharomyces pombe. Preferably, a dicarboxylic acid transporterprotein is a malic acid transporter protein (MAE) which has at least 80,85, 90, 95 or 99% sequence identity with SEQ ID NO: 36. Preferably theMAE comprises SEQ ID NO: 36. Surprisingly, it was found that aeukaryotic cell according to the present invention further comprising adicarboxylic acid transporter, such as a malic acid transporter asdescribed herein produced an increased amount of succinic acid ascompared to a eukaryote cell not comprising a dicarboxylic acidtransporter protein.

The present invention also relates to the use of a dicarboxylic acidtransporter, preferably a malic acid transporter protein, in aeukaryotic cell to increase succinic acid production. Preferably, themalic acid transporter is derived from Schizosaccharomyces pombe.

In a preferred embodiment a eukaryotic cell according to the presentinvention is a yeast comprising nucleotide sequences encoding aNAD(H)-dependent fumarate reductase, a malate dehydrogenase, aheterologous fumarase, a heterologous PEP carboxykinase and aheterologous dicarboxylic acid transporter and overexpresses a pyruvatecarboxylase (PYC), as described, including the preferred embodiments,herein above. Surprisingly, it found that a yeast of the inventioncomprising the nucleotide sequences encoding the enzymes as describedherein produced an increased amount of succinic acid as compared to ayeast comprising either of the nucleotide sequences alone.

In another preferred embodiment a eukaryotic cell according to thepresent invention comprises reduced activity of enzymes that convertNAD(H) to NAD⁺ compared to the activity of these enzymes in a wild-typecell.

Preferably, the cell according to the present invention is a cellwherein at least one gene encoding alcohol dehydrogenase is notfunctional. An alcohol dehydrogenase gene that is not functional is usedherein to describe a eukaryotic cell which comprises a reduced alcoholdehydrogenase activity compared to a cell wherein all genes encoding analcohol dehydrogenase are functional. A gene may become not functionalby known methods in the art, for instance by mutation, disruption, ordeletion, for instance by the method disclosed by Gueldener et. al.2002, Nucleic Acids Research, Vol. 30, No. 6, e23. Preferably, aeukaryotic cell is a yeast cell such as Saccharomyces cerevisiae,wherein one or more genes adh1 and/or adh2, encoding alcoholdehydrogenase are inactivated.

Preferably, the cell according to the present invention furthercomprises at least one gene encoding glycerol-3-phosphate dehydrogenasewhich is not functional. A glycerol-3-phosphate dehydrogenase gene thatis not functional is used herein to describe a eukaryotic cell, whichcomprises a reduced glycerol-3-phosphate dehydrogenase activity, forinstance by mutation, disruption, or deletion of the gene encodingglycerol-3-phosphate dehydrogenase, resulting in a decreased formationof glycerol as compared to the wild-type cell. Surprisingly, it wasfound that the eukaryotic cell comprising reduced alcohol dehydrogenaseactivity and/or glycerol-3-phosphate dehydrogenase activity and aNAD(H)-dependent fumarase resulted in an increased production ofsuccinic acid as compared to a cell wherein one or more gene(s) encodingalcohol dehydrogenase and/or glycerol-3-phosphate dehydrogenase are notinactivated.

The present invention also relates to a process for the production ofsuccinic acid comprising fermenting a eukaryotic cell comprising atleast one gene encoding alcohol dehydrogenase is not functional and / orat least one gene encoding glycerol-3-phosphate dehydrogenase which isnot functional.

In another preferred embodiment the recombinant eukaryotic cellaccording to the present invention comprises at least one gene encodingsuccinate dehydrogenase that is not functional. A succinatedehydrogenase that is not functional is used herein to describe aeukaryotic cell, which comprises a reduced succinate dehydrogenaseactivity by mutation, disruption, or deletion, of at least one geneencoding succinate dehydrogenase resulting in a increased formation ofsuccinic acid as compared to the wild-type cell. A eukaryotic cellcomprising a gene encoding succinate dehydrogenase that is notfunctional may for instance be Aspergillus niger, preferably anAspergillus niger, wherein one or more genes encoding succinatedehydrogenase, such as sdhA and sdhB is/are not functional, for instanceby deletion of these genes.

Preferably, a eukaryotic cell according to the invention is a yeast,preferably Saccharomyces cerevisiae, preferably a Saccharomycescerevisiae comprising one or more of the nucleotide sequences selectedfrom SEQ ID NO: 9 and SEQ ID NO: 10. A eukaryotic cell according to thepresent invention may also be a filamentous fungus, preferably A. niger,preferably A. niger comprising one or more nucleotide sequences selectedfrom SEQ ID NO: 7 and SEQ ID NO: 8.

Preferably, a eukaryotic cell according to the present inventioncomprising any one of the genetic modifications described herein iscapable of producing at least 0.3, 0.5, 0.7, g/L succinic acid,preferably at least 1 g/L succinic acid, preferably at least 1.5preferably at least 2, or 2.5, 4.5 preferably at least 8, 10, 15, or 20g / L succinic acid but usually below 200 or below 150 g / L.

A preferred eukaryotic cell according to the present invention may beable to grow on any suitable carbon source known in the art and convertit to succinic acid. The eukaryotic cell may be able to convert directlyplant biomass, celluloses, hemicelluloses, pectines, rhamnose,galactose, fucose, maltose, maltodextrines, ribose, ribulose, or starch,starch derivatives, sucrose, lactose and glycerol. Hence, a preferredhost organism expresses enzymes such as cellulases (endocellulases andexocellulases) and hemicellulases (e.g. endo- and exo-xylanases,arabinases) necessary for the conversion of cellulose into glucosemonomers and hemicellulose into xylose and arabinose monomers,pectinases able to convert pectines into glucuronic acid andgalacturonic acid or amylases to convert starch into glucose monomers.Preferably, the cell is able to convert a carbon source selected fromthe group consisting of glucose, fructose, galactose, xylose, arabinose,sucrose, raffinose, lactose and glycerol.

In another aspect, the present invention relates to a process for thepreparation of succinic acid, comprising fermenting the eukaryotic cellaccording to the present invention, wherein succinic acid is prepared.

It was found advantageous to use a eukaryotic cell according to theinvention in the process for the production of succinic acid, becausemost eukaryotic cells do not require sterile conditions for propagationand are insensitive to bacteriophage infections.

Preferably, the succinic acid that is prepared in the process accordingto the present invention is further converted into a desirable product.A desirable product may for instance be a polymer, such as polybutylenesuccinic acid (PBS), a deicing agent, or a surfactant.

The process according to the present invention may be run under aerobicand anaerobic conditions. Preferably, the process is carried out underanaerobic conditions or under micro-aerophilic or oxygen limitedconditions. An anaerobic fermentation process is herein defined as afermentation process run in the absence of oxygen or in whichsubstantially no oxygen is consumed, preferably less than 5, 2.5 or 1mmol/L/h, and wherein organic molecules serve as both electron donor andelectron acceptors.

An oxygen-limited fermentation process is a process in which the oxygenconsumption is limited by the oxygen transfer from the gas to theliquid. The degree of oxygen limitation is determined by the amount andcomposition of the ingoing gasflow as well as the actual mixing/masstransfer properties of the fermentation equipment used.

Preferably, in a process under oxygen-limited conditions, the rate ofoxygen consumption is at least 5.5, more preferably at least 6 and evenmore preferably at least 7 mmol/L/h.

The process for the production of succinic acid according to the presentinvention may be carried out at any suitable pH between 1 and 9.Preferably, the pH in the fermentation broth is between 2 and 7,preferably between 3 and 5. It was found advantageous to be able tocarry out the process according to the present invention at a low pH,since this prevents bacterial contamination. In addition, since the pHdrops during succinic acid production, a lower amount of titrant may beneeded to keep the pH at a desired level.

A suitable temperature at which the process according to the presentinvention may be carried out is between 5 and 60° C., preferably between10 and 50° C., more preferably between 15 and 35° C., more preferablybetween 18° C. and 30° C. The skilled man in the art knows which optimaltemperatures are suitable for fermenting a specific eukaryotic cell.

Preferably, succinic acid is recovered from the fermentation broth by asuitable method known in the art, for instance by crystallisation andammonium precipitation.

Preferably, the succinic acid that is prepared in the process accordingto the present invention is further converted into a pharmaceutical,cosmetic, food, feed, or chemical product. Succinic acid may be furtherconverted into a polymer, such as polybutylene succinate (PBS) or othersuitable polymers derived therefrom.

The present invention also relates to a fermentation broth comprising asuccinic acid obtainable by a process according to the presentinvention.

The invention relates to a process for the production of succinic acidby a yeast or a filamentous fungus as succinic acid producer, wherebyfumarate reductase from Trypanosoma brucei is used to increase succinicacid production, wherein preferably the fumarate reductase is active inthe cytosol.

Genetic Modifications

Standard genetic techniques, such as overexpression of enzymes in thehost cells, genetic modification of host cells, or hybridisationtechniques, are known methods in the art, such as described in Sambrookand Russel (2001) “Molecular Cloning: A Laboratory Manual (3^(rd)edition), Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, or F. Ausubel et al, eds., “Current protocols in molecularbiology”, Green Publishing and Wiley Interscience, New York (1987).Methods for transformation, genetic modification etc of fungal hostcells are known from e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 andWO 00/37671, W090/14423, EP-A-0481008, EP-A-0635 574 and U.S. Pat. No.6,265,186.

The following examples are for illustrative purposes only and are not tobe construed as limiting the invention.

DESCRIPTION OF THE FIGURES

FIG. 1. Map of the pGBTOP-11 vector used for expression of fumaratereductase in A. niger

FIG. 2: Plasmid map of pGBS414SUS-07, encoding mitochondrial fumaratereductase m1 (FRDm1) from Trypanosoma brucei for expression inSaccharomyces cerevisiae. CPO denotes codon pair optimized.

FIG. 3: Plasmid map of pGBS414SUS-08, encoding glycosomal fumaratereductase (FRDg) from Trypanosoma brucei for expression in Saccharomycescerevisiae. CPO denotes codon pair optimized.

FIG. 4: Plasmid map of pDEL-SDHA

FIG. 5: Map of plasmid pGBTPAn1, for overexpression FRDm1 in A. niger.

FIG. 6: Replacement scheme of sdhA

FIG. 7: Plasmid map of pGBS416FRD-1, encoding mitochondrial fumaratereductase m1 (FRDm1) from Trypanosoma brucei for expression inSaccharomyces cerevisiae. CPO denotes codon pair optimized.

FIG. 8: Plasmid map of pGBS416FRE-1, encoding glycosomal fumaratereductase (FRDg) from Trypanosoma brucei for expression in Saccharomycescerevisiae. CPO denotes codon pair optimized.

FIG. 9: Plasmid map of pGBS414PPK-1, containing PEP carboxykinase fromActinobacillus succinogenes (PCKa) for expression in Saccharomycescerevisiae. The synthetic gene construct TDH1 promoter-PCKa-TDH1terminator was cloned into expression vector pRS414. CPO denotes codonpair optimized.

FIG. 10: Plasmid map of pGBS414PPK-2, containing PEP carboxykinase fromActinobacillus succinogenes (PCKa) and mitochondrial fumarate reductasem1 from Trypanosoma brucei (FRDm1) for expression in Saccharomycescerevisiae. The synthetic gene constructs TDH1 promoter-PCKa-TDH1terminator and TDH3 promoter-FRDm1-TDH3 terminator were cloned intoexpression vector pRS414. CPO denotes codon pair optimized.

FIG. 11: Plasmid map of pGBS414PPK-3, containing PEP carboxykinase fromActinobacillus succinogenes (PCKa) and glycosomal fumarate reductasefrom Trypanosoma brucei (FRDg) for expression in Saccharomycescerevisiae. The synthetic gene constructs TDH1 promoter-PCKa-TDH1terminator and TDH3 promoter-FRDg-TDH3 terminator were cloned intoexpression vector pRS414. CPO denotes codon pair optimized.

FIG. 12: Plasmid map of pGBS414PEK-1, containing PEP carboxykinase fromMannheimia succiniciproducens (PCKm) for expression in Saccharomycescerevisiae. The synthetic gene construct TDH1 promoter-PCKm-TDH1terminator was cloned into expression vector pRS414. CPO denotes codonpair optimized.

FIG. 13: Plasmid map of pGBS414PEK-2, containing PEP carboxykinase fromMannheimia succiniciproducens (PCKm) and mitochondrial fumaratereductase m1 from Trypanosoma brucei (FRDm1) for expression inSaccharomyces cerevisiae. The synthetic gene constructs TDH1promoter-PCKm-TDH1 terminator and TDH3 promoter-FRDm1-TDH3 terminatorwere cloned into expression vector pRS414. CPO denotes codon pairoptimized.

FIG. 14: Plasmid map of pGBS414PEK-3, containing PEP carboxykinase fromMannheimia succiniciproducens (PCKm) and glycosomal fumarate reductasefrom Trypanosoma brucei (FRDg) for expression in Saccharomycescerevisiae. The synthetic gene constructs TDH1 promoter-PCKm-TDH1terminator and TDH3 promoter-FRDg-TDH3 terminator were cloned intoexpression vector pRS414. CPO denotes codon pair optimized.

FIG. 15: Plasmid map of pGBS415FUM-2, containing fumarase from Rhizopusoryzae (FUMR) and cytoplasmic malate dehydrogenase from Saccharomycescerevisiae truncated for the first 12 amino acids (delta12N MDH2) forexpression in Saccharomyces cerevisiae. The synthetic gene constructsTDH1 promoter-FUMR-TDH1 terminator and DH3 promoter-MDH3-TDH3 terminatorwere cloned into expression vector pRS415. CPO denotes codon pairoptimized.

FIG. 16: Plasmid map of pGBS415FUM-3, containing fumarase from Rhizopusoryzae (FUMR) and peroxisomal malate dehydrogenase from Saccharomycescerevisiae (MDH3) for expression in Saccharomyces cerevisiae. Thesynthetic gene constructs TDH1 promoter-FUMR-TDH1 terminator and TDH3promoter-MDH3-TDH3 terminator were cloned into expression vector pRS415.CPO denotes codon pair optimized.

FIG. 17: Succinic acid levels in strains SUC-101 (◯, empty vectorscontrol), SUC-148 (▪, overexpression of PCKa, MDH3, FUMR, FRDm1),SUC-149 (□, PCKa, MDH3, FUMR, FRDg), SUC-150 (♦, PCKm, MDH3, FUMR,FRDm1), SUC-151 (⋄, PCKm, MDH3, FUMR, FRDg), SUC-152 (, PCKa, MDH3,FUMR), SUC-154 (×, PCKm, MDH3, FUMR) and SUC-169 (▴, PCKm, delta12NMDH2,FUMR, FRDm1). All overexpressed genes were codon pair optimized forexpression in S. cerevisiae. All data represent averages of 3independent growth experiments of SUC-148, 149, 150, 151, 152, 154 andSUC-169 and averages of 6 independent growth experiments of SUC-101.

FIG. 18: Plasmid map of pGBS416MAE-1, containing malate permease fromSchizosaccharomyces pombe (SpMAE1) for expression in Saccharomycescerevisiae. The synthetic gene construct EnoI promoter-MAE1-Eno1terminator was cloned into expression vector pRS416. CPO denotes codonpair optimized.

FIG. 19: Succinic acid levels in strains SUC-101 (◯, empty vectorscontrol), SUC-169 (▴, PCKm, delta12NMDH2, FUMR, FRDm1) and SUC-194 (▪,PCKm, delta12NMDH2, FUMR, FRDm1, SpMAE1). All overexpressed genes werecodon pair optimized for expression in S. cerevisiae. All data representaverages of 3 independent growth experiments of SUC-169 and SUC-194 andaverages of 6 independent growth experiments of SUC-101.

FIG. 20: Succinic acid levels in strains SUC-103 (◯, adh1/2 and gpd1deletion mutant; empty vectors control), SUC-201 (□, adh1/2 and gpd1deletion mutant; PCKa, MDH3, FUMR, FRDg) and SUC-200 (▪, adh1/2 and gpd1deletion mutant; PCKa, MDH3, FUMR, FRDg, SpMAE1). All overexpressedgenes were codon pair optimized for expression in S. cerevisiae.

FIG. 21: Plasmid map of pGBS426PYC-2, containing pyruvate carboxylasefrom Saccharomyces cerevisiae for expression in Saccharomycescerevisiae. The PYC2 coding nucleotide sequence was obtained by PCRusing genomic DNA from strain CEN.PK113-5D as template and the PCRproduct was cloned into expression vector p426GPD.

FIG. 22: Plasmid map of pGBS414FRE-1, encoding glycosomal fumaratereductase (FRDg) from Trypanosoma brucei for expression in Saccharomycescerevisiae. The synthetic gene construct TDH3 promoter-FRDg-TDH3terminator was cloned into expression vector pRS414.

FIG. 23: Succinic acid levels in strains SUC-226 (□, PCKa, MDH3, FUMR,FRDg), -227 (▴, PYC2, PCKa, MDH3, FUMR, FRDg), SUC-228 (▪, PYC2, MDH3,FUMR, FRDg) and SUC-230 (◯, MDH3, FUMR, FRDg). Data represents theaverage of 3 independent growth experiments.

EXAMPLES Example 1 Cloning of Fumarate Reductases from TrypanosomaBrucei in Aspergillus Niger 1.1. Expression Constructs

Mitochondrial fumarate reductase ml (FRDm1) [E.C. 1.3.1.6], GenBankaccession number 60460035, from Trypanosoma brucei was analysed for thepresence of signal sequences using SignalP 3.0(http://www.cbs.dtu.dk/services/SignalP/) Bendtsen, J. et al. (2004)Mol. Biol., 340:783-795 and TargetP 1.1(http://www.cbs.dtu.dk/services/TargetP/) Emanuelsson, O. et al. (2007)Nature Protocols 2, 953-971. A putative mitochondrial targeting sequencein the N-terminal half of the protein was identified, including apossible cleavage site between pos. 25 and 26 (D-S).

It was shown that FRDm1 recombinant protein lacking the 68 N-terminalresidues, relocalized to the cytosol of the procyclic trypanosomes(Coustou et al., J Biol Chem. 2005 Apr. 29; 280(17):16559-70). Theseresults indicate that the predicted N-terminal signal motif of FRDm1 isrequired for targeting to the mitochondrion. The first 68 amino acidswere removed from SEQ ID NO: 1 (corresponding to nucleotide sequence SEQID NO: 2) and a new methionine amino acid was reintroduced, whichresulted in SEQ ID NO: 3. SEQ ID NO: 3 was subjected to the codon-pairmethod as disclosed in WO2008/000632 for A. niger. The resultingsequence SEQ ID NO: 7 was put behind the constitutive GPDA promotersequence SEQ ID NO: 11, wherein the last 10 nucleotide sequences werereplaced with optimal Kozak sequence CACCGTAAA. Convenient restrictionsites were added. The stop codon TAA in SEQ: ID NO: 7 was modified toTAAA. The resulting sequence was synthesised at Sloning (Puchheim,Germany). The fragment was SnaBI, SfiI cloned in the A. niger expressionvector pGBTOP11 (FIG. 1) using appropriate restriction sites. Theresulting plasmid comprising FRDm1 was named pGBTOPAn1 (FIG. 5).

Likewise, glycosomal fumarate reductase (FRDg) [E.C. 1.3.1.6], GenBankaccession number 23928422, from Trypanosoma brucei was analysed forperoxisomal targeting in filamentous fungi using the PTS1 predictorhttp://mendel.imp.ac.at/mendeljsp/sat/pts1/PTS1predictor.jsp with thefungi-specific prediction function. The C-terminal amino acids atposition 1140-1142 (SKI) were removed from the protein SEQ ID NO: 4(corresponding to nucleotide sequence SEQ ID NO: 5), resulting in SEQ IDNO: 6. SEQ ID NO: 6, was subjected to the codon-pair method as disclosedin PCT/EP2007/05594 for A. niger. The stop codon TAA in SEQ ID NO: 8 wasmodified to TAAA. The resulting sequence SEQ ID NO: 8 was put behind theconstitutive GPDA promoter sequence SEQ ID NO: 11, and convenientrestriction sites were added. The resulting sequence was synthesised atSloning (Puchheim, Germany). The fragment was SnaBI, SfiI cloned in theA. niger expression vector pGBTOP11 (FIG. 1) using appropriaterestriction sites.

1.2. Transformation of A. Niger

A. niger WT-1: This A. niger strain is CBS513.88 comprising deletions ofthe genes encoding glucoamylase (glaA), fungal amylase and acid amylase.A. niger WT 1 was constructed by using the “MARKER-GENE FREE” approachas described in EP 0 635 574 B1.

The expression constructs are co-transformed to strain A. niger WT-1according to the method described by Tilburn, J. et al. (1983) Gene 26,205-221 and Kelly, J. & Hynes, M. (1985) EMBO J., 4, 475-479 with thefollowing modifications:

Spores are germinated and cultivated for 16 hours at 30 degrees Celsiusin a shake flask placed in a rotary shaker at 300 rpm in Aspergillusminimal medium (100 ml). Aspergillus minimal medium contains per litre:6 g NaNO₃, 0.52 g KCl, 1.52 g KH₂PO₄, 1.12 ml 4 M KOH, 0.52 gMgSO₄.7H₂O, 10 g glucose, 1 g casaminoacids, 22 mg ZnSO₄.7H₂O, 11 mgH₃BO₃, 5 mg FeSO₄.7H₂O, 1.7 mg CoCl₂.6H₂O, 1.6 mg CuSO₄.5H₂O, 5 mgMnCl₂.2H₂O, 1.5 mg Na₂MoO₄.2H₂O, 50 mg EDTA, 2 mg riboflavin, 2 mgthiamine-HCl, 2 mg nicotinamide, 1 mg pyridoxine-HCL, 0.2 mg panthotenicacid, 4 g biotin, 10 ml Penicillin (5000 IU/ml) Streptomycin (5000UG/ml) solution (Gibco).

Novozym 234™ (Novo Industries) instead of helicase is used for thepreparation of protoplasts;

After protoplast formation (60-90 minutes), KC buffer (0.8 M KCl, 9.5 mMcitric acid, pH 6.2) is added to a final volume of 45 ml, the protoplastsuspension is centrifuged for 10 minutes at 3000 rpm at 4 degreesCelsius in a swinging-bucket rotor. The protoplasts are resuspended in20 ml KC buffer and subsequently 25 ml of STC buffer (1.2 M sorbitol, 10mM Tris-HCl pH 7.5, 50 mM CaCl₂) is added. The protoplast suspension iscentrifuged for 10 minutes at 3000 rpm at 4 degrees Celsius in aswinging-bucket rotor, washed in STC-buffer and resuspended inSTC-buffer at a concentration of 10E8 protoplasts/ml;

To 200 microliter of the protoplast suspension, the DNA fragment,dissolved in 10 microliter TE buffer (10 mM Tris-HCl pH 7.5, 0.1 mMEDTA) and 100 microliter of PEG solution (20% PEG 4000 (Merck), 0.8 Msorbitol, 10 mM Tris-HCl pH 7.5, 50 mM CaCl₂) is added;

After incubation of the DNA-protoplast suspension for 10 minutes at roomtemperature, 1.5 ml PEG solution (60% PEG 4000 (Merck), 10 mM Tris-HClpH 7.5, 50 mM CaCl₂) is added slowly, with repeated mixing of the tubes.After incubation for 20 minutes at room temperature, suspensions arediluted with 5 ml 1.2 M sorbitol, mixed by inversion and centrifuged for10 minutes at 4000 rpm at room temperature. The protoplasts areresuspended gently in 1 ml 1.2 M sorbitol and plated onto solidselective regeneration medium consisting of either Aspergillus minimalmedium without riboflavin, thiamine.HCL, nicotinamide, pyridoxine,panthotenic acid, biotin, casaminoacids and glucose. In case ofacetamide selection the medium contains 10 mM acetamide as the solenitrogen source and 1 M sucrose as osmoticum and C-source.Alternatively, protoplasts are plated onto PDA (Potato Dextrose Agar,Oxoid) supplemented with 1-50 microgram/ml phleomycin and 1M sucrose asosmosticum. Regeneration plates are solidified using 2% agar (agar No.1, Oxoid L11). After incubation for 6-10 days at 30 degrees Celsius,conidiospores of transformants are transferred to plates consisting ofAspergillus selective medium (minimal medium containing acetamide assole nitrogen source in the case of acetamide selection or PDAsupplemented with 1-50 microgram/ml phleomycin in the case of phleomycinselection) with 2% glucose and 1.5% agarose (Invitrogen) and incubatedfor 5-10 days at 30 degrees Celsius. Single transformants are isolatedand this selective purification step is repeated once upon whichpurified transformants are stored.

1.3. Shake Flask Growth of A. Niger

In total 10 transformants are selected for each construct and thepresence of the construct is confirmed by PCR using primers specific forthe constructs. Subsequently spores are inoculated in 100 ml Aspergillusminimal enriched medium comprising 100 g/l glucose. Strains are grown inan incubator at 250 rotations per minute for four days at 34 degreesCelsius. The supernatant of the culture medium is analysed for oxalicacid, malic acid, fumaric acid and succinic acid formation by HPLC andcompared to a non transformed strain.

1.4 HPLC Analysis

HPLC is performed for the determination of organic acids and sugars indifferent kinds of samples. The principle of the separation on aPhenomenex Rezex-RHM-Monosaccharide column is based on size exclusion,ion-exclusion and ion-exchange using reversed phase mechanisms.Detection takes place by differential refractive index and ultra violetdetectors.

Example 2A Cloning of Fumarate Reductases from Trypanosoma Brucei inSaccharomyces Cerevisiae 2A.1. Expression Constructs

Mitochondrial fumarate reductase m1 (FRDm1) [E.C. 1.3.1.6], GenBankaccession number 60460035, from Trypanosoma brucei was analysed for thepresence of signal sequences and codon optimized as described in section1.1 for expression in S. cerevisiae. The resulting sequence SEQ ID NO: 9was put behind the constitutive TDH3 promoter sequence SEQ ID NO: 12 andbefore the TDH3 terminator sequence SEQ ID NO: 13, and convenientrestriction sites were added. The stop codon TGA in SEQ ID NO: 9 wasmodified to TAAG. The resulting sequence was synthesised at Sloning(Puchheim, Germany). The expression construct pGBS414SUS-07 was createdafter a BamHI/NotI restriction of the S. cerevisiae expression vectorpRS414 (Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27) andsubsequently ligating in this vector a BamHI/NotI restriction fragmentconsisting of the fumarate reductase synthetic gene construct (FIG. 2).The ligation mix is used for transformation of E. coli DH10B(Invitrogen) resulting in the yeast expression construct pGBS414SUS-07(FIG. 2).

Likewise, glycosomal fumarate reductase (FRDg) [E.C. 1.3.1.6], GenBankaccession number 23928422, from Trypanosoma brucei was analysed forperoxisomal targeting and codon optimisation was applied as described insection 1.1 for expression in S. cerevisiae. The resulting sequence SEQID NO: 10 was put behind the constitutive TDH3 promoter sequence SEQ IDNO: 12 and before the TDH3 terminator sequence SEQ ID NO: 13, andconvenient restriction sites were added. The stop codon TGA in SEQ IDNO: 10 was modified to TAAG. The resulting sequence was synthesised atSloning (Puchheim, Germany). The expression construct pGBS414SUS-08 wascreated after a BamHI/NotI restriction of the S. cerevisiae expressionvector pRS414 (Sirkoski R. S. and Hieter P, Genetics, 1989,122(1):19-27) and subsequently ligating in this vector a BamHI/NotIrestriction fragment consisting of the fumarate reductase synthetic geneconstruct (FIG. 3). The ligation mix is used for transformation of E.coli DH10B (Invitrogen) resulting in the yeast expression constructpGBS414SUS-08 (FIG. 3).

The constructs pGBS414SUS-07 and pGBS414SUS-08 are independentlytransformed into S. cerevisiae strains CEN.PK113-6B (MATA ura3-52leu2-112 trp1-289), RWB066 (MATA ura3-52 leu2-112 trp1-289 adh1::loxadh2::Kanlox) and RWB064 (MATA ura3-52 leu2-112 trp1-289 adh1::loxadh2::lox gpd1::Kanlox). Transformation mixtures are plated on YeastNitrogen Base (YNB) w/o AA (Difco)+2% glucose supplemented withappropriate amino acids. Transformants are inoculated in Verduyn mediumcomprising glucose supplemented with appropriate amino acids (Verduyn etal., 1992, Yeast. July; 8(7):501-17) and grown under aerobic, anaerobicand oxygen-limited conditions in shake flasks. The medium for anaerobiccultivation is supplemented with 0.01 g/l ergosterol and 0.42 g/l Tween80 dissolved in ethanol (Andreasen and Stier, 1953, J. cell. Physiol,41, 23-36; Andreasen and Stier, 1954, J. Cell. Physiol, 43: 271-281).All yeast cultures are grown at 30° C. in a shaking incubator at 250-280rpm. At different incubation times, aliquots of the cultures areremoved, centrifuged and the medium is analysed by HPLC for formation ofoxalic acid, malic acid, fumaric acid and succinic acid as described insection 1.4.

Example 2B Cloning of Fumarate Reductases from Trypanosoma Brucei inSaccharomyces Cerevisiae 28.1. Expression Constructs

In a similar way as disclosed in Example 2A.1. mitochondrial fumaratereductase from Trypanosoma brucei (FRDm, SEQ ID NO: 9) was ligated in aS. cerevisiae expression vector pRS416 (Sirkoski R. S. and Hieter P,Genetics, 1989, 122(1):19-27). The ligation mix was used fortransformation of E. coli TOP10 cells (Invitrogen) resulting in theyeast expression constructs and pGBS416FRD-1 (FIG. 7).

Likewise, glycosomal fumarate reductase (FRDg, SEQ ID NO: 10) fromTrypanosoma brucei was ligated in an S. cerevisiae expression vectorpRS416. The ligation mix was used for transformation of E. coli TOP10cells (Invitrogen) resulting in the yeast expression constructpGBS416FRE-1 (FIG. 8).

28.2. Transformation and Microtiterplates (MTP's) Growth Experiments

The constructs pGBS416FRD-1 and pGBS416FRE-1 were independentlytransformed into S. cerevisiae strain CEN.PK113-5D (MATA ura3-52). Asnegative control, empty vector pRS416 was transformed into strain CEN.PK113-5D. Transformation mixtures were plated on Yeast Nitrogen Base (YNB)w/o AA (Difco)+2% glucose. The following numbers of individualtransformants were inoculated in duplo in 250 microlitres Verduyn mediumcomprising 2% glucose in 96 deep-well MTP's and pre-cultured at 30degrees Celsius, 550 rpm, and a humidity of 80% in an Infors Microplateshaking incubator: 12 pGBS416FRD-1 (FRDm1), 12 pGBS416FRE-1 (FRDg) and24 pRS416 empty vector control transformants. After 3 days, 25microlitres of the pre-culture present in the wells of the MTP plateswas transferred to new 96 deep-well MTP's containing Verduyn mediumcontaining glucose and CaCO₃ (end-concentrations: glucose 10%, CaCO3 1%w/v in a total volume of 250 microlitres). After 3 and 7 days of growthat 30° C., 550 rpm, and a humidity of 80% in an Infors Microplateshaking incubator, the MTP's were centrifuged for 2 minutes at 2000 rpm,and 200 microliters of supernatant was harvested using the Multimek 96(Beckman). The supernatant was analyzed by HPLC as described in Example1.4 for the presence succinic acid. The results are shown in Table 1.

TABLE 1 Effect of introduction of mitochondrial (FRDm1) and glycosomalfumarate reductase (FRDg) from T. brucei in S. cerevisiae on thesuccinic acid production levels after 3 and 7 days of incubation S.cerevisiae comprising Succinic acid (mg/l) Succinic acid (mg/l) plasmid:after 3 days after 7 days Empty vector pRS416 138 ± 18 (n = 48) 203 ± 48(n = 48) pGBS416FRD-1 (FRDm1) 340 ± 65 (n = 24) 399 ± 72 (n = 24)pGBS416FRE-1 (FRDg) 489 ± 30 (n = 24) 516 ± 57 (n = 24)

The results in Table 1 show that introduction and overexpression ofmitochondrial fumarate reductase (FRDm1) from T. brucei resulted inincreased succinic acid production levels (2.47 fold, p=6.96E-14,Student's t-test, after 3 days incubation and 1.97 fold, p=8.63E-14,Student's t-test after 7 days incubation).

Likewise, introduction and overexpression of glycosomal fumaratereductase (FRDg) from T. brucei resulted in increased succinic acidproduction levels (3.55 fold, p=5.08E-32, Student's t-test, after 3 daysincubation and a 2.55 fold increase, p=8.63E-25, Student's t-test after7 days incubation).

Example 2C Expression of PEP Carboxykinase from ActinobacillusSuccinogenes or Mannheimia Succiniciproducens and Malate Dehydrogenasefrom Saccharomyces Cerevisiae and Fumarase from Rhizopus Oryzae andFumarate Reductase from Trypanosoma Brucei in Saccharomyces Cerevisiae2C.1 Gene Sequences Phosphoenolpyruvate Carboxykinase:

Phosphoenolpyruvate carboxykinase [E.C. 4.1.1.49], Gen Bank accessionnumber 152977907, from Actinobacillus succinogenes was analysed for thepresence of signal sequences using SignalP 3.0(http://www.cbs.dtu.dk/services/SignalP/) Bendtsen, J. et al. (2004)Mol. Biol., 340:783-795 and TargetP 1.1(http://www.cbs.dtu.dk/services/TargetP/) Emanuelsson, O. et al. (2007)Nature Protocols 2, 953-971. Analysis as described by Schluter et al.,(2007) NAR, 35, D815-D822 revealed a putative PTS2 signal sequence atposition 115-123. The A. succinogenes sequence was modified to resemblethe Mannheimia succiniciproducens protein sequence by replacing theamino acids EGY at position 120-122 with DAF resulting in amino acidsequence SEQ ID NO: 14 (nucleotide sequence SEQ ID NO: 15). SEQ ID NO:14 was subjected to the codon-pair method as disclosed in WO2008/000632for S. cerevisiae. The stop codon TAA in the resulting nucleotidesequence SEQ ID NO: 16 was modified to TAAG. This SEQ ID NO: 16containing stop codon TAAG was put behind the constitutive TDH1 promotersequence SEQ ID NO: 25 and before the TDH1 terminator sequence SEQ IDNO: 26, and convenient restriction sites were added. The resultingsequence SEQ ID NO: 29 was synthesised at Sloning (Puchheim, Germany).

Likewise phosphoenolpyruvate carboxykinase [E.C. 4.1.1.49], GenBankaccession number 52426348, from Mannheimia succiniciproducens wasanalysed for the presence of signal sequences as described in Schluteret al., (2007) NAR, 35, D815-D822. The sequence as shown in SEQ ID NO:17 required no modifications. SEQ ID NO: 17 was subjected to thecodon-pair method as disclosed in WO2008/000632 for S. cerevisiae. Thestop codon TAA in the resulting sequence SEQ ID NO: 18 was modified toTAAG. SEQ ID NO: 18 containing stop codon TAAG was put behind theconstitutive TDH1 promoter sequence SEQ ID NO: 25 and before the TDH1terminator sequence SEQ ID NO: 26. Convenient restriction sites wereadded. The resulting synthetic construct (SEQ ID NO: 30) was synthesisedat Sloning (Puchheim, Germany).

Malate Dehydrogenase

Cytoplasmic malate dehydrogenase (Mdh2p) [E.C. 1.1.1.37], GenBankaccession number 171915, is regulated by carbon catabolite repression:transcription of MDH2 is repressed and Mdh2p is degraded upon additionof glucose to glucose-starved cells. Mdh2p deleted for the 12amino-terminal amino acids is less-susceptible for glucose-induceddegradation (Minard and McAlister-Henn, J Biol Chem. 1992 Aug. 25;267(24):17458-64). To avoid glucose-induced degradation of Mdh2, thenucleotides encoding the first 12 amino acids were removed, and a newmethionine amino acid was introduced (SEQ ID NO: 19) for overexpressionof Mdh2 in S. cerevisiae. SEQ ID NO: 19 was subjected to the codon-pairmethod as disclosed in WO2008/000632 for S. cerevisiae. The stop codonTAA in the resulting in SEQ ID NO: 20, was modified to TAAG. SEQ ID NO:20 containing a modified stop codon TAAG, encoding delta12NMDH2, was putbehind the constitutive TDH3 promoter sequence SEQ ID NO: 12 and beforethe TDH3 terminator sequence SEQ ID NO: 13, and convenient restrictionsites were added. The resulting synthetic construct (SEQ ID NO: 31) wassynthesised at Sloning (Puchheim, Germany).

Peroxisomal malate dehydrogenase (Mdh3p) [E.C. 1.1.1.37], GenBankaccession number 1431095, was analysed for peroxisomal targeting infilamentous fungi using the PTS1 predictorhttp://mendel.imp.ac.at/mendeljsp/sat/pts1/PTS1predictor.jsp with thefungi-specific prediction function. The C-terminal amino acids atposition 341-343 (SKL) were removed from protein MDH3 resulting in SEQID NO: 21. SEQ ID NO: 21 was subjected to the codon-pair method asdisclosed in WO2008/000632 for S. cerevisiae. The stop codon TGA in theresulting sequence SEQ ID NO: 22 was modified to TAAG. SEQ ID NO: 22containing TAAG as stop codon was synthesized behind the constitutiveTDH3 promoter sequence SEQ ID NO: 27 (600 by upstream of start codon)and before the TDH3 terminator sequence SEQ ID NO: 28 (300 by downstramof stop codon), and convenient restriction sites were added. Theresulting sequence SEQ ID NO: 32 was synthesised at Sloning (Puchheim,Germany).

Fumarase:

Fumarase [E.C. 4.2.1.2], GenBank accession number 469103, from Rhizopusoryzae (FumR) was analysed for the presence of signal sequences usingSignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) Bendtsen, J. etal. (2004) Mol. Biol., 340:783-795 and TargetP 1.1(http://www.cbs.dtu.dk/services/TargetP/) Emanuelsson, O. et al. (2007)Nature Protocols 2, 953-971. A putative mitochondrial targeting sequencein the first 23 amino acid of the protein was identified. To avoidpotential targeting to mitochondria in S. cerevisiae, the first 23 aminoacids were removed from FumR and a methionine amino acid wasreintroduced resulting in SEQ ID NO: 23. SEQ ID NO: 23 was subjected tothe codon-pair method as disclosed in WO2008/000632 for S. cerevisiaeresulting in SEQ ID NO: 24. The stop codon TAA in SEQ ID NO: 24 wasmodified to TAAG. SEQ ID NO: 24 containing TAAG as stop codon wassynthesized behind the constitutive TDH1 promoter sequence SEQ ID NO: 25and before the TDH1 terminator sequence SEQ ID NO: 26 and convenientrestriction sites were added. The resulting synthetic construct SEQ IDNO: 33 was synthesised at Sloning (Puchheim, Germany).

Fumarate Reductase:

Gene sequences of mitochondrial fumarate reductase (FRDm1) andglycosomal fumarate reductase (FRDg) from T. brucei were designed andsynthesized as described under 2A.1.

2C.2. Construction of Expression Constructs

The expression constructs pGBS414PPK-1 (FIG. 9), pGBS414PPK-2 (FIG. 10)and pGBS414PPK-3 (FIG. 11) were created after a BamHI/NotI restrictionof the S. cerevisiae expression vector pRS414 (Sirkoski R. S. and HieterP, Genetics, 1989, 122(1):19-27) and subsequently ligating in thisvector a BamHI/NotI restriction fragment consisting of thephosphoenolpyruvate carboxykinase (origin Actinobacillus succinogenes)synthetic gene construct (SEQ ID NO: 29). The ligation mix was used fortransformation of E. coli TOP10 (Invitrogen) resulting in the yeastexpression construct pGBS414PPK-1. Subsequently, pGBK414PPK-1 wasrestricted with AscI and NotI. To create pGBS414PPK-2, an AscI/NotIrestriction fragment consisting of mitochondrial fumarate reductase fromT. brucei (FRDm1) synthetic gene construct (SEQ ID NO: 34) was ligatedinto the restricted pGBS414PPK-1 vector. The ligation mix was used fortransformation of E. coli TOP10 (Invitrogen) resulting in the yeastexpression construct pGBS414PPK-2 (FIG. 10). To create pGBS414PPK-3, anAscI/NotI restriction fragment consisting of glycosomal fumaratereductase from T. brucei (FRDg) synthetic gene construct (SEQ ID NO: 35)was ligated into the restricted pGBS414PPK-1 vector. The ligation mixwas used for transformation of E. coli TOP10 (Invitrogen) resulting inthe yeast expression construct pGBS414PPK-3 (FIG. 11).

The expression constructs pGBS414PEK-1 (FIG. 12), pGBS414PEK-2 (FIG. 13)and pGBS414PEK-3 (FIG. 14) were created after a BamHI/NotI restrictionof the S. cerevisiae expression vector pRS414 (Sirkoski R. S. and HieterP, Genetics, 1989, 122(1):19-27) and subsequently ligating in thisvector a BamHI/NotI restriction fragment consisting of thephosphoenolpyruvate carboxykinase (origin Mannheimia succiniciproducens)synthetic gene construct (SEQ ID NO: 30). The ligation mix was used fortransformation of E. coli TOP10 (Invitrogen) resulting in the yeastexpression construct pGBS414PEK-1. Subsequently, pGBK414PEK-1 wasrestricted with AscI and NotI. To create pGBS414PEK-2, an AscI/NotIrestriction fragment consisting of mitochondrial fumarate reductase fromT. brucei (FRDm1) synthetic gene construct (SEQ ID NO: 34) was ligatedinto the restricted pGBS414PEK-1 vector. The ligation mix was used fortransformation of E. coli TOP10 (Invitrogen) resulting in the yeastexpression construct pGBS414PEK-2 (FIG. 13). To create pGBS414PEK-3, anAscI/NotI restriction fragment consisting of glycosomal fumaratereductase from T. brucei (FRDg) synthetic gene construct (SEQ ID NO: 35)was ligated into the restricted pGBS414PEK-1 vector. The ligation mixwas used for transformation of E. coli TOP10 (Invitrogen) resulting inthe yeast expression construct pGBS414PEK-3 (FIG. 14).

The expression constructs pGBS415FUM-2 (FIG. 15) and pGBS415FUM-3 (FIG.16) were created after a BamHI/NotI restriction of the S. cerevisiaeexpression vector pRS415 (Sirkoski R. S. and Hieter P, Genetics, 1989,122(1):19-27) and subsequently ligating in this vector a BamHI/NotIrestriction fragment consisting of the fumarase (origin Rhizopus oryzae)synthetic gene construct (SEQ ID NO: 33). The ligation mix was used fortransformation of E. coli TOP10 (Invitrogen) resulting in the yeastexpression construct pGBS415FUM-1. Subsequently, pGBK415FUM-1 wasrestricted with AscI and NotI. To create pGBS415FUM-2, an AscI/NotIrestriction fragment consisting of cytoplasmic malate dehydrogenase fromS. cerevisiae (delta12N MDH2) synthetic gene construct (SEQ ID NO: 31)was ligated into the restricted pGBS415FUM-1 vector. The ligation mixwas used for transformation of E. coli TOP10 (Invitrogen) resulting inthe yeast expression construct pGBS415FUM-2 (FIG. 15). To createpGBS415FUM-3, an AscI/NotI restriction fragment consisting ofperoxisomal malate dehydrogenase from S. cerevisiae (MDH3) syntheticgene construct (SEQ ID NO: 32) was ligated into the restrictedpGBS415FUM-1 vector. The ligation mix was used for transformation of E.coli TOP10 (Invitrogen) resulting in the yeast expression constructpGBS415FUM-3 (FIG. 16).

2C.3. S. Cerevisiae Strains

Different combinations of plasmids pGBS414PPK-1, pGBS414 PPK-2,pGBS414PPK-3, pGBS414PEK-1, pGBS414PEK-2, pGBS414PEK-3, pGBS415FUM-2,pGBS415-FUM-3 were transformed into S. cerevisiae strain CEN.PK113-6B(MATA ura3-52 leu2-112 trp1-289), resulting in the yeast strainsdepicted in Table 2. In addition to the mentioned plasmids, pRS416(empty vector) was transformed to create prototrophic yeast strains. Theexpression vectors were transformed into yeast by electroporation. Thetransformation mixtures were plated on Yeast Nitrogen Base (YNB) w/o AA(Difco)+2% glucose.

TABLE 2 Yeast strains constructed for Example 2C. Name BackgroundPlasmids Genes SUC-148 CEN.PK113-6B pGBS414PPK-2 PCKa, FRDm1pGBS415FUM-3 FUMR, MDH3 pRS416 (empty vector) SUC-149 CEN.PK113-6BpGBS414PPK-3 PCKa, FRDg pGBS415FUM-3 FUMR, MDH3 pRS416 (empty vector)SUC-150 CEN.PK113-6B pGBS414PEK-2 PCKm, FRDm1 pGBS415FUM-3 FUMR, MDH3pRS416 (empty vector) SUC-151 CEN.PK113-6B pGBS414PEK-3 PCKm, FRDgpGBS415FUM-3 FUMR, MDH3 pRS416 (empty vector) SUC-152 CEN.PK113-6BpGBS414PPK-1 PCKa pGBS415FUM-3 FUMR, MDH3 pRS416 (empty vector) SUC-154CEN.PK113-6B pGBS414PEK-1 PCKm pGBS415FUM-3 FUMR, MDH3 pRS416 (emptyvector) SUC-169 CEN.PK113-6B pGBS414PEK-2 PCKm, FRDm1 pGBS415FUM-2 FUMR,Δ12NMDH2 pRS416 (empty vector) SUC-101 CEN.PK113-6B pRS414 (emptyvector) pRS415 (empty vector) pRS415 (empty vector)

2C.4. Growth Experiments and Succinic Acid Production

Transformants were inoculated in 20 ml pre-culture medium consisting ofVerduyn medium (Verduyn et al., 1992, Yeast. July; 8(7):501-17)comprising 2% galactose (w/v) and grown under aerobic conditions in 100ml shake flasks in a shaking incubator at 30° C. at 250 rpm. After 72hours, the culture was centrifuged for 5 minutes at 4750 rpm. 1 mlsupernatant was used to measure succinic acid levels by HPLC asdescribed in section 1.4. The remaining supernatant was decanted and thepellet (cells) was resuspended in 1 ml production medium. The productionmedium consisted of Verduyn medium with 10% galactose (w/v) and 1% CaCO3(w/v). The resuspended cells were inoculated in 50 ml production mediumin 100 ml shake flasks and grown in a shaking incubator at 30° C. at 100rpm. At various time points, 1 ml sample was taken from the culturesuccinic acid levels were measured by HPLC as described in section 1.4(FIG. 17).

Strains transformed with empty vectors (control strain) produced up to0.3 g/L succinic acid. Overexpression of PEP carboxykinase from M.succiniciproducens (PCKm), peroxisomal malate dehydrogenase (MDH3) fromS. cerevisiae and fumarase from R. oryzae (FUMR) resulted in productionof 0.9 g/L succinic acid production. Overexpression of PEP carboxykinasefrom A. succinogenes (PCKa), MDH3 and FUMR resulted in a slight increasein succinic acid production to 1.0 g/L.

These results show that in S. cerevisiae as decribed increased succinicacid production about 3 times.

Additional overexpression of mitochondrial fumarate reductase (FRDm1)from T. brucei further increased succinic acid production levels;overexpression of PCKa, MDH3, FUMR, FRDm1 resulted in production of 2.6g/L succinic acid, and overexpression of PCKm, MDH3, FUMR and FRDm1resulted in production of 2.7 g/L succinic acid. Overexpression ofdelta12NMDH2 in combination with PCKm, FUMR and FRDm1 resulted inproduction of 2.7 g/L succinic acid, indicating that similar levels ofsuccinic acid were produced using either truncated MDH2 or MDH3.Additional overexpression of glycosomal fumarate reductase (FRDg) fromT. brucei resulted in an even higher increase in succinic acidproduction levels; overexpression of PCKa, MDH3, FUMR and FRDg resultedin production of 3.9 g/L succinic acid, whereas overexexpression ofPCKm, MDH3, FUMR and FRDg resulted in slightly lower production of 3.6g/L succinic acid.

The results show addition of NAD(H) dependent fumarate reductase asdisclosed herein in a S. cerevisiae comprising a genetic modification ofPCKa/m, MDH3 and FUMR significantly increased succinic acid productionlevels.

Overexpression of FRDg had a more positive effect on succinic acidproduction levels in S. cerevisiae compared to overexpression of FRDm1in S. cerevisiae.

Example 2D Effect of Overexpression of a Dicarboxylic Acid Transporteron Succinic Acid Production in Succinic Acid Producing S. CerevisiaeCells 2D.1. Gene Sequences

Malate permease, GenBank accession number 119368831, fromSchizosaccharomyces pombe (SEQ ID NO: 36) was subjected to thecodon-pair method as disclosed in WO2008/000632 for S. cerevisiaeresulting in SEQ ID NO: 37. The stop codon TAA in SEQ ID NO: 37 wasmodified to TAAG. SEQ ID NO: 37 containing TAAG as stop codon was putbehind the constitutive ENO1 promoter sequence SEQ ID NO: 38 and beforethe ENO1 terminator sequence SEQ ID NO: 39, and convenient restrictionsites were added. In the ENO1 promotor, T at position 596 (−5) waschanged to A in order to obtain a better Kozak sequence. The resultingsequence SEQ ID NO: 40 was synthesised at Sloning (Puchheim, Germany).

2D.2. Construction of Expression Constructs

The expression constructs pGBS416MAE-1 (FIG. 18) was created after aBamHI/NotI restriction of the S. cerevisiae expression vector pRS416(Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27) andsubsequently ligating in this vector a BamHI/NotI restriction fragmentconsisting of the Schizosaccharomyces pombe malate transporter syntheticgene construct (SEQ ID NO: 40). The ligation mix was used fortransformation of E. coli TOP10 (Invitrogen) resulting in the yeastexpression construct pGBS416MAE-1.

2D.3. S. Cerevisiae Strains

Plasmids pGBS414PEK-2, pGBS415FUM-2 and pGBS416MAE-1 (described under2C.2.) were transformed into S. cerevisiae strain CEN.PK113-6B (MATAura3-52 leu2-112 trp1-289) to create strain SUC-194, overexpressingPCKm, delta12NMDH2, FUMR, FRDm1 and SpMAE1. All genes were codon pairoptimized for expression in S. cerevisiae.

The expression vectors were transformed into yeast by electroporation.The transformation mixtures were plated on Yeast Nitrogen Base (YNB) w/oAA (Difco)+2% glucose. Strains SUC-101 is described in Table 2.

TABLE 3 Yeast strains constructed for Example 2D. Name BackgroundPlasmids Genes SUC-132 CEN.PK113-6B pGBS414PEK-2 PCKm, FRDm1pGBS415FUM-2 FUMR, Δ12NMDH2 pRS416 (empty vector) SUC-194 CEN.PK113-6BpGBS414PEK-2 PCKm, FRDm1 pGBS415FUM-2 FUMR, Δ12NMDH2 pRS416MAE-1 SpMAE1

2D.4. Growth Experiments and Succinic Acid Production in Wildtype CEN.PKStrains

Growth parameters and sample analysis were performed as described underexample 2C.4 with the following modifications: pre-culturing wasperformed using 2% glucose (w/v) as carbon source. In the productionmedium 10% glucose (w/v) was used as carbon source.

Strains transformed with empty vectors (control strain) produced up to0.3 g/L succinic acid. Additional overexpression of SpMAE1 in strainSUC-194, overexpressing PCKm, delta12NMDH2, FUMR and FRDm1 resulted inincreased succinic acid production levels to 4.6 g/L, whereas strainSUC-132, overexpressing PCKm, delta12NMDH2, FUMR and FRDm1 resulted inproduction of 2.7 g/L succinic acid.

The results show that insertion of a malate transporter in a S.cerevisiae comprising the genetic modifications as described hereinfurther increased succinic acid production at least 1.5 times.

Example 2E Effect of a Dicarboxylic Acid Transporter in S. CerevisiaeComprising a Deletion of the Genes Alcohol Dehydrogenase 1 and 2 (adh1,adh2) and the gene glycerol-3-phosphate dehydrogenase 1 (gpd1) onSuccinic Acid Production Levels 2E.1. Gene Sequences

Described under 2D.1.

2E.2. Construction of Expression Constructs

Described under 2D.2.

2E.3. S. Cerevisiae Strains

Plasmids pGBS414PPK-3, pGBS415FUM-3 and pGBS416MAE-1 (described under2C.2.) were transformed into S. cerevisiae strain RWB064 (MA TA ura3-52leu2-112 trp1-289 adh1::lox adh2::lox gpd1::Kanlox) to create strainSUC-201, overexpressing PCKa, MDH3, FUMR, FRDg and SpMAE1. All geneswere codon pair optimized for expression in S. cerevisiae.

TABLE 4 Yeast strains constructed for Example 2E. Name BackgroundPlasmids Genes SUC-200 CEN.PK113-6B pGBS414PPK-3 PCKa, FRDg adh1::loxadh2::lox pGBS415FUM-3 FUMR, MDH3 gpd1::Kanlox pGBS416MAE-1 SpMAE1SUC-201 CEN.PK113-6B pGBS414PPK-3 PCKa, FRDg adh1::lox adh2::loxpGBS415FUM-3 FUMR, MDH3 gpd1::Kanlox pRS416 (empty vector) SUC-103CEN.PK113-6B pRS414 (empty vector) adh1::lox adh2::lox pRS415 (emptyvector) gpd1::Kanlox pRS415 (empty vector)2E.4. Growth Experiments and Succinic Acid Production in CEN.PK StrainsDeleted for the Genes Alcohol Dehydrogenase 1 and 2 (Adh1, Adh2) and theGene glycerol-3-phosphate dehydrogenase 1 (gpd1)

Growth parameters and sample analysis were performed as described underexample 2C.4 with the following modifications: pre-culturing wasperformed using 2% galactose (w/v) as carbon source. 5% galactose (w/v)was added to the production medium at t=0, 3 and 7 days.

Strain SUC-103 transformed with empty vectors (control strain) produced0.9 g/L succinic acid after growth for 10 days in production medium(FIG. 20). Overexpression of PCKa, MDH3, FUMR and FRDg in strain RWB064resulted in increased succinic acid production levels to 2.5 g/L (strainSUC-201, FIG. 20). Additional overexpression of SpMAE1 besides PCKa,MDH3, FUMR and FRDg in strain RWB064 resulted in a further increase ofsuccinic acid production levels to 11.9 g/L (strain SUC-200, FIG. 20).

The results show that overexpression of a malate transporter in S.cerevisiea comprising a deletion of alcohol dehydrogenase andglycerol-3-phosphate dehydrogenase genes resulted in a significantincrease in succinic acid production levels. In addition it was shownthat deletion of the gene adh1, adh2 and gpd1 (SUC 103) resulted inincreased succinic acid production levels as compare to a wild typestrain (SUC 101, Table 2).

Example 2F Cloning of Phosphoenolpyruvate Carboxykinase fromActinobacillus Succinogenes, Pyruvate Carboxylase from SaccharomycesCerevisiae, Malate Dehydrogenase from Saccharomyces Cerevisiae, Fumarasefrom Rhizopus Oryzae in Saccharomyces Cerevisiae and Fumarate Reductasefrom Trypanosoma Brucei 2F.1. Gene Sequences

Gene sequences of PEP carboxykinase from A. succinogenes, malatedehydrogenase from S. cerevisiae, fumarase from R. oryzae and fumaratereductase from T. brucei are described under 2F.1. Cytoplasmic pyruvatecarboxylase from Saccharomyces cerevisiae (Pyc2p) [E.C. 6.4.1.1.],GenBank accession number 1041734, SEQ ID NO: 41, is encoded by thenucleotide sequence SEQ ID NO: 42. Genomic DNA from S. cerevisiae strainCEN.PK113-5D (MATA ura3-52) was used as template to amplify the PYC2coding sequence (SEQ ID NO: 42), using primers P1 (SEQ ID NO: 43) and P2(SEQ ID NO: 44), and the Phusion DNA polymerase (Finnzymes, Finland)according to manufacturer's instructions. Convenient restriction siteswere included in the primers for further cloning purposes.

2F.2. Construction of Expression Constructs

The expression construct pGBS426PYC-2 (FIG. 21) was created after aSpeI/XhoI restriction of the S. cerevisiae expression vector p426GPD(Mumberg et al., Gene. 1995 Apr. 14; 156(1):119-22) and subsequentlyligating in this vector a SpeI/XhoI restriction fragment consisting ofthe amplified PYC2 nucleotide sequence (SEQ ID NO: 42). The ligation mixwas used for transformation of E. coli TOP10 (Invitrogen) resulting inthe yeast expression construct pGBS426PYC-2 (FIG. 21). Construction ofexpression vectors pGBS414PPK-3 and pGBS415FUM-3 is described under2C.2. Expression construct pGBS414FRE-1 was created after a BamHI/NotIrestriction of the S. cerevisiae expression vector pRS414 (Sirkoski R.S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligatingin this vector a BamHI/NotI restriction fragment consisting of theglycosomal fumarate reductase (origin Trypanosoma brucei) synthetic geneconstruct (SEQ ID NO: 35). The ligation mix was used for transformationof E. coli TOP10 (Invitrogen) resulting in the yeast expressionconstruct pGBS414FRE-1 (FIG. 22).

2F.3. S. Cerevisiae Strains

Strains SUC-226, SUC-227, SUC-228 and SUC-230 were obtained bytransformation of different combinations of the plasmids pGBS414FRE-1,pGBS414PPK-3, pGBS415FUM-1, pGBS426PYC-2 and p426GPD into strainCEN.PK113-6B (MATA ura3-52 leu2-112 trp1-289), as depicted in Table 5.

TABLE 5 Yeast strains constructed for Example 2F. Name BackgroundPlasmids Genes SUC-226 CEN.PK113-6B pGBS414PPK-3 PCKa, FRDg pGBS415FUM-3FUMR, MDH3 p426GPD (empty vector) SUC-227 CEN.PK113-6B pGBS414PPK-3PCKa, FRDg pGBS415FUM-3 FUMR, MDH3 pGBS426PYC-2 PYC2 SUC-228CEN.PK113-6B pGBS414FRE-1 FRDg pGBS415FUM-3 FUMR, MDH3 pGBS426PYC-2 PYC2SUC-230 CEN.PK113-6B pGBS414FRE-1 FRDg pGBS415FUM-3 FUMR, MDH3 p426GPD(empty vector)

2F.4. Growth Experiments and Succinic Acid Production

Growth parameters and sample analysis were performed as described underexample 2C.4 with the following modifications: pre-culturing wasperformed using 2% glucose (w/v) as carbon source. In the productionmedium 10% glucose (w/v) was used as carbon source.

As depicted in FIG. 23 strain SUC-230, overexpressing MDH3, FUMR andFRDg, produced up to 3.0 g/L succinic acid. Additional overexpression ofPCKa increased succinic acid production up to 3.4 g/L (strain SUC-226),and additional overexpression of PYC2 increased succinic acid productionup to 3.7 g/L (strain SUC-228). Surprisingly, overexpression of bothPCKa and PYC2 (SUC-227) resulted in 1.5 increase of succinic acidproduction levels up to 5.0 g/L, as compared to the effect of PCK andPYC alone. These results show a synergistic effect of combinedoverexpression of both PEP carboxykinase from A. succinogenes (PCKa) andpyruvate carboxylase from S. cerevisiae (PYC2) on succinic acidproduction levels in S. cerevisiae.

Example 3 Inactivation of Succinate Dehydrogenase Encoding Genes inAspergillus Niger 3.1. Identification

Genomic DNA of Aspergillus niger strain CBS513.88 was sequenced andanalyzed. Two genes with translated proteins annotated as homologues tosuccinate dehydrogenase proteins were identified and named sdhA and sdhBrespectively. Sequences of the sdhA (An16g07150) and sdhB (An02g12770)loci are available on genbank with accession numbers 145253004 and145234071 respectively. Gene replacement vectors for sdhA and sdhB weredesigned according to known principles and constructed according toroutine cloning procedures (see FIG. 6). The vectors compriseapproximately 1000 by flanking regions of the sdh ORFs for homologousrecombination at the predestined genomic loci. In addition, they containthe A. nidulans bi-directional amdS selection marker driven by the gpdApromoter, in-between direct repeats. The general design of thesedeletion vectors were previously described in EP635574B and WO 98/46772.

3.2. Inactivation of the SdhA Gene in Aspergillus Niger.

Linear DNA of deletion vector pDEL-SDHA (FIG. 4) was isolated and usedto transform Aspergillus niger CBS513.88 as described in: Biotechnologyof Filamentous fungi: Technology and Products. (1992) Reed Publishing(USA); Chapter 6: Transformation p. 113 to 156. This linear DNA canintegrate into the genome at the sdhA locus, thus substituting the sdhAgene by the amdS gene as depicted in FIG. 6. Transformants were selectedon acetamide media and colony purified according to standard proceduresas described in EP635574B. Spores were plated on fluoro-acetamide mediato select strains, which lost the amdS marker. Growing colonies werediagnosed by PCR for integration at the sdhA locus and candidate strainstested by Southern analyses for deletion of the sdhA gene. Deletion ofthe sdhA gene was detectable by the ˜2.2 kb size reduction of DNAfragments (4.6 kb wild-type fragment versus 2.4 kb for a successfuldeletion of SDHA) covering the entire locus and hybridized toappropriate probes. Approximately 9 strains showed a removal of thegenomic sdhA gene from a pool of approximately 96 initial transformants.

Strain dSDHA was selected as a representative strain with the sdhA geneinactivated. The succinic acid production of dSDHA was determined inmicrotiter plates as described in Example 4.

Example 4 Cloning of FRDm from Trypanosoma Brucei in Aspergillus NigerdSDHA

A. niger strain dSDHA of example 3.2. was transformed with theexpression construct pGBTOPAn1 (FIG. 5) comprising truncatedmitochondrial fumarate reductase m1 (FRDm1, SEQ ID NO:7) as described inExample 1.1. E. coli DNA was removed by NotI digestion. A. nigertransformants were picked using Qpix and transferred onto MTP'scontaining Aspergillus selective media. After 7 days incubation at 30degrees Celsius the biomass was transferred to microtiter plates (MTP's)containing PDA by hand or colony picker. After 7 days incubation at 30degrees Celsius, the biomass was sporulated. These spores wereresuspended using the Multimek 96 (Beckman) in 100 microlitres minimalenriched Aspergillus medium containing 10% glucose. Subsequently 2 MTPwith 170 micolitres minimal enriched Aspergillus medium containing 10%glucose and 1% CaCO3 were inoculated with 30 microlitres of the sporesuspension. Likewise, A. niger strains dSDHA and CBS513.88 wereinoculated in the MTP's. These MTP's were incubated for 5 days at 34degrees Celsius80% humidity. After 5 days 160 microlitres were harvestedusing the Multimek 96 (Beckman) and succinic acid was determined by HPLCas described in Example 1.4. The results are shown in Table 6.

TABLE 6 Effect of deletion of succinate dehydrogenase (SDHA) andinsertion of mitochondrial fumarate reductase (FRDm1) from T. brucei inA. niger on succinic acid production levels. A. niger strain Succinicacid mg/l CBS513.88 38 dSDHA 50 dSDHA + gGBTOPAn1 583  (FRDm1)

Table 6 clearly shows an increased production of succinic acid by A.niger that comprises mitochondrial fumarate reductase from T. brucei

1. A recombinant eukaryotic cell selected from the group consisting of ayeast and a filamentous fungus comprising a nucleotide sequence encodinga NAD(H)-dependent fumarate reductase that catalyses the conversion offumaric acid to succinic acid.
 2. A cell according to claim 1, whereinthe cell expresses a nucleotide sequence encoding an enzyme thatcatalyses the formation of succinic acid, wherein the nucleotidesequence encodes a NAD(H)-dependent fumarate reductase, comprising anamino acid sequence that has at least 40% sequence identity with theamino acid sequence of SEQ ID NO:1, and/or SEQ ID NO: 3, and/or SEQ IDNO:4, and/or SEQ ID NO: 6;
 3. A cell according to claim 1, wherein theNAD(H)-dependent fumarate reductase is derived from a Trypanosoma sp. 4.A cell according to claim 1, wherein the NAD(H)-dependent fumaratereductase is active in the cytosol upon expression of the nucleotidesequence encoding NAD(H)-dependent fumarate reductase.
 5. A cellaccording to claim 1, wherein the cell overexpresses a nucleotidesequence encoding a pyruvate carboxylase.
 6. A cell according to claim1, further comprising a nucleotide sequence encoding a heterologousphosphoenolpyruvate carboxykinase.
 7. A cell according to claim 1,further comprising a nucleotide sequence encoding a malate dehydrogenaseactive in the cytosol upon expression of the nucleotide sequenceencoding malate dehydrogenase.
 8. A cell according to claim 1, furthercomprising a nucleotide sequence encoding an enzyme that catalyses theconversion of malic acid to fumaric acid in the cytosol, upon expressionof the nucleotide sequence encoding enzyme that catalyses the conversionof malic acid to fumaric acid.
 9. A cell according to claim 1, furthercomprising a nucleotide sequence encoding a dicarboxylic acidtransporter.
 10. A cell according to claim 1, wherein at least one geneencoding alcohol dehydrogenase is not functional.
 11. A cell accordingto claim 1, wherein at least one gene encoding glycerol-3-phosphatedehydrogenase is not functional.
 12. A cell according to claim 1,wherein at least one gene encoding succinate dehydrogenase is notfunctional.
 13. A cell according to claim 1, which is an Aspergillus,preferably an Aspergillus niger.
 14. A cell according to claim 1, whichis a Saccharomyces cerevisiae
 15. A process for the preparation ofsuccinic acid, comprising fermenting a eukaryotic cell according toclaim 1, in a suitable fermentation medium, wherein succinic acid isprepared.
 16. A process according to claim 15, wherein the succinic acidprepared is used for the production of a pharmaceutical, cosmetic, food,feed or chemical product
 17. A fermentation broth comprising succinicacid obtainable by the process according to claim 16.